In response to RFA-DA-12-009, our multidisciplinary team proposes to study the correlates, consequences, and mechanisms of increased immune activation associated with injection drug use (IDU). Key to this proposal are preliminary data that we generated during a pilot study conducted between June 2009 and September 2011. We discovered increased levels of immune activation systemically and in gut- associated lymphoid tissue (GALT) in HIV-1 uninfected IDUs. Our findings in tissue are novel and though immune activation in IDUs has been reported, an understanding of correlates and mechanism has not been addressed. Our broad term goals are to use a multifaceted approach including systems biology to explore the following questions; 1) What are the mechanisms of increased immune activation in blood and tissue associated with active IDU? 2) Can we separate the effects of 3 possible contributors to increased levels of immune activation- non-sterile injection, direct effects of opiates and chronic infection with Hepatitis C? 3) Is the heightened immune activation associated with active IDU reversible when injection ceases, and if so, what are the dynamics of the reversibility? 4) How does active injection impact immune reconstitution in HIV-1 infected IDUs on combination antiretroviral therapy (cART) and can we characterize differences qualitatively and quantitatively? To answer these complex questions we have devised a strategy to recruit a cohort of HIV- 1-uninfected and -infected active IDUs along with very carefully matched appropriate controls from whom we will collect blood, tissue, and behavioral data both cross-sectionally and longitudinally. HIV-1-infected active injectors will be treated with cART and monitored closely for virologic and immunologic responses and compared to appropriate non-injecting controls. We will measure levels of cellular activation and proliferation using markers such as CD38 and HLA-DR and Ki67 respectively on subsets of cells from blood and tissue. Levels of soluble markers of immune activation will also be measured in patient plasma. Behavioral data including drug use behaviors will be examined as correlates of immune activation and reconstitution. In response to the RFA and reflecting the most innovative aspect of this project, we will use a systems biology approach to address the issues above. We will sort specific cell populations from peripheral blood and tissue for transcriptional profiling of mRNA with the use of microarrays. In addition we will also collect cells from in vitro experiments for transcriptional profiling to study the effects of the addition and withdrawal of select opiates on specific T cell subsets. With the assistance of the Data Analysis Core established by The Rockefeller University Center for Clinical and Translational Science, we will analyze the transcriptional profiling data to identify gene signatures and pathways for correlation with markers of immune activation, immune reconstitution, and injecting behaviors. By studying the intersection of IDU and infection with HIV-1 and HCV, we aim to improve treatment outcomes in affected populations and individuals.